Considerable experimental evidence now supports a critically important pathophysiological role for the calcium-activated, protein-degradative enzyme calpain in the secondary injury process that follows traumatic brain injury (TBI). This has suggested that that early treatment with a calpain-inhibiting drug should be able to reduce post-TBI secondary injury to brain tissue and thus facilitate neurological recovery and survival. Accordingly, several small molecule calpain inhibitors have been discovered. A few of these have been tested in rodent TBI models and have shown some promise in terms of either a decrease in calpain- mediated proteolytic damage in the injured brain tissue or an improvement in neurological recovery. However, none of the available calpain inhibitors have been systematically studies in TBI models so that translation of their effects into clinical development would be possible. Thus, the overall goal of this project is to carefully examine the ability of 5 carefully selected calpain inhibitors, including three newer compounds, to be able to effectively inhibit post-traumatic cytoskeletal proteoytic degradation, mitochondrial dysfunction and neurodegeneration and to improve neurological recovery. The 5 compounds will first be screened for their ability to inhibit calpain activation in normal mice in a dose-related fashion (Aim 1). The most effective compounds will be advanced into testing for their cytoskeletal (Aim 2), mitochondrial (Aim 3) and neuroprotective effects (Aim 4) in a diffuse and a focal TBI model. One of the 5 compounds selected is a dual inhibitor of calpain proteolytic degradation and free radical-induced lipid peroxidation. The latter secondary injury process is known to enhance calpain activation by causing impairment of neuronal calcium homeostatic mechanisms. As a result, the increase in intracellular calcium exacerbates calpain activation. Therefore, a dual inhibitor or calpain and lipid peroxidation would be expected to be more effective than a either type of compound alone. In addition to looking at the dose-related benefits of early administration of a calpain inhibitor or a dual calpain inhibitor/lipid peroxidation inhibitor in two different TBI models, the therapeutic efficacy window and the optimum duration of treatment will be explored. The resulting data will address the overall hypothesis that calpain inhibition is a practical neuroprotective therapeutic approach and define how best to design anti-calpain therapy so that future clinical trials in TBI patients are enabled.